Amy L. Vincent

Chapter 3 Swine Influenza Viruses: A North American Perspective

Influenza is a zoonotic viral disease that represents a health and economic threat to both humans and animals worldwide. Swine influenza (SI) was first recognized clinically in pigs in the Midwestern U.S., in 1918, coinciding with the human influenza pandemic known as the Spanish flu. Since that time SI has remained of importance to the swine industry throughout the world. In this review, the epidemiology of swine influenza virus (SIV) infection in North American pigs is described in detail. The first 80 years of SI remained relatively static, whereas the last decade has become dynamic with the establishment of many emerging subtypes. With the increasing number of novel subtypes and genetic variants, the control of SI has become increasingly difficult and innovative strategies to combat this economically important zoonotic disease are critical. Therefore, protective immune responses against influenza virus infections as well as new paradigms of vaccine development in pigs are discussed in the review. It is expected that the dynamic evolutionary changes of SIVs in North American pigs will continue, making currently available prophylactic approaches of limited use to control the spread and economic losses associated with this important swine pathogen.

Detection of two porcine circovirus type 2 genotypic groups in United States swine herds

Summary.In late 2005, sporadic cases of an acute onset disease of high mortality were observed in 10- to 16-week-old growing pigs among several swine herds of the United States. Tissues from the affected pigs in Kansas, Iowa, and North Carolina were examined, and porcine circovirus type 2 (PCV2) was detected consistently among these tissues. Phylogenetically, PCV2 can be divided into two major genotypic groups, PCV2-group 1 and PCV2-group 2. Whereas PCV2-group 1 isolates were detected in all the diseased animals, only two of the diseased animals harbored PCV2-group 2 isolates. This observation is important because PCV2-group 1 isolates had never been reported in the United States before (GenBank as of May 16, 2006), and they are closely related to the PCV2-group 1 isolates that have been described in Europe and Asia, previously. Our analysis revealed that each genotypic group contains a distinct stretch of nucleotide or amino acid sequence that may serve as a signature motif for PCV2-group 1 or PCV2-group 2 isolates.

Identification of H2N3 influenza A viruses from swine in the United States

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian α2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.

Vaccination of Pigs against Swine Influenza Viruses by Using an NS1-Truncated Modified Live-Virus Vaccine

ABSTRACT Swine influenza viruses (SIV) naturally infect pigs and can be transmitted to humans. In the pig, genetic reassortment to create novel influenza subtypes by mixing avian, human, and swine influenza viruses is possible. An SIV vaccine inducing cross-protective immunity between different subtypes and strains circulating in pigs is highly desirable. Previously, we have shown that an H3N2 SIV (A/swine/Texas/4199-2/98 [TX98]) containing a deleted NS1 gene expressing a truncated NS1 protein of 126 amino acids, NS1▴126, was attenuated in swine. In this study, 4-week-old pigs were vaccinated with the TX98 NS1▴126 modified live virus (MLV). Ten days after boosting, pigs were challenged with wild-type homologous H3N2 or heterosubtypic H1N1 SIV and sacrificed 5 days later. The MLV was highly attenuated and completely protected against challenge with the homologous virus. Vaccinated pigs challenged with the heterosubtypic H1N1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated H1N1 control pigs. Remarkably, vaccinated pigs challenged with the H1N1 SIV had significantly lower microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, H1N1-challenged pigs. All vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin A antibodies against H3N2 SIV antigens. Vaccinated pigs were seronegative for NS1, indicating the potential use of the TX98 NS1▴126 MLV as a vaccine to differentiate infected from vaccinated animals.

Characterization of a newly emerged genetic cluster of H1N1 and H1N2 swine influenza virus in the United States

H1 influenza A viruses that were distinct from the classical swine H1 lineage were identified in pigs in Canada in 2003–2004; antigenic and genetic characterization identified the hemagglutinin (HA) as human H1 lineage. The viruses identified in Canadian pigs were human lineage in entirety or double (human–swine) reassortants. Here, we report the whole genome sequence analysis of four human-like H1 viruses isolated from U.S. swine in 2005 and 2007. All four isolates were characterized as triple reassortants with an internal gene constellation similar to contemporary U.S. swine influenza virus (SIV), with HA and neuraminidase (NA) most similar to human influenza virus lineages. A 2007 human-like H1N1 was evaluated in a pathogenesis and transmission model and compared to a 2004 reassortant H1N1 SIV isolate with swine lineage HA and NA. The 2007 isolate induced disease typical of influenza virus and was transmitted to contact pigs; however, the kinetics and magnitude differed from the 2004 H1N1 SIV. This study indicates that the human-like H1 SIV can efficiently replicate and transmit in the swine host and now co-circulates with contemporary SIVs as a distinct genetic cluster of H1 SIV.

Review of influenza A virus in swine worldwide: a call for increased surveillance and research.

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high‐priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.

The role of swine in the generation of novel influenza viruses.

The ecology of influenza A viruses is very complicated involving multiple host species and viral genes. Avian species have variable susceptibility to influenza A viruses with wild aquatic birds being the reservoir for this group of pathogens. Occasionally, influenza A viruses are transmitted to mammals from avian species, which can lead to the development of human pandemic strains by direct or indirect transmission to man. Because swine are also susceptible to infection with avian and human influenza viruses, genetic reassortment between these viruses and/or swine influenza viruses can occur. The potential to generate novel influenza viruses has resulted in swine being labelled ‘mixing vessels’. The mixing vessel theory is one mechanism by which unique viruses can be transmitted from an avian reservoir to man. Although swine can generate novel influenza viruses capable of infecting man, at present, it is difficult to predict which viruses, if any, will cause a human pandemic. Clearly, the ecology of influenza A viruses is dynamic and can impact human health, companion animals, as well as the health of livestock and poultry for production of valuable protein commodities. For these reasons, influenza is, and will continue to be, a serious threat to the wellbeing of mankind.

Vaccine-Induced Anti-HA2 Antibodies Promote Virus Fusion and Enhance Influenza Virus Respiratory Disease

Heterologous influenza A vaccination–induced antibodies correlated with vaccine-associated enhanced respiratory disease. The (Mis)match Game Even the most beneficial things—like vaccines—sometimes have a downside. Learning what causes the downside is critical for avoiding it. In the case of viral vaccines, there have been some reports of rare vaccine-induced disease enhancement—for example, vaccine-associated enhanced respiratory disease (VAERD) for influenza. Khurana et al. now report that mismatched strains of the same subtype of influenza may lead to VAERD in pigs. The authors vaccinated pigs with whole inactivated H1N2 influenza virus. These pigs had enhanced pneumonia and disease after infection with another strain—pH1N1. Looking more closely, the authors found that the immune sera from the H1N2-vaccinated pigs contained high titers of cross-reactive hemagglutinin antibodies. These antibodies actually enhanced pH1N1 infection in cell culture by promoting virus membrane fusion activity, and this enhanced fusion correlated with lung pathology. This mechanism of VAERD should be considered when devising strategies to devise a universal flu vaccine. Vaccine-induced disease enhancement has been described in connection with several viral vaccines in animal models and in humans. We investigated a swine model to evaluate mismatched influenza vaccine-associated enhanced respiratory disease (VAERD) after pH1N1 infection. Vaccinating pigs with whole inactivated H1N2 (human-like) virus vaccine (WIV-H1N2) resulted in enhanced pneumonia and disease after pH1N1 infection. WIV-H1N2 immune sera contained high titers of cross-reactive anti-pH1N1 hemagglutinin (HA) antibodies that bound exclusively to the HA2 domain but not to the HA1 globular head. No hemagglutination inhibition titers against pH1N1 (challenge virus) were measured. Epitope mapping using phage display library identified the immunodominant epitope recognized by WIV-H1N2 immune sera as amino acids 32 to 77 of pH1N1-HA2 domain, close to the fusion peptide. These cross-reactive anti-HA2 antibodies enhanced pH1N1 infection of Madin-Darby canine kidney cells by promoting virus membrane fusion activity. The enhanced fusion activity correlated with lung pathology in pigs. This study suggests a role for fusion-enhancing anti-HA2 antibodies in VAERD, in the absence of receptor-blocking virus-neutralizing antibodies. These findings should be considered during the evaluation of universal influenza vaccines designed to elicit HA2 stem-targeting antibodies.

Characterization of an influenza A virus isolated from pigs during an outbreak of respiratory disease in swine and people during a county fair in the United States

In August 2007, pigs and people became clinically affected by an influenza-like illness during attendance at an Ohio county fair. Influenza A virus was identified from pigs and people, and the virus isolates were characterized as swine H1N1 similar to swine viruses currently circulating in the U.S. pig population. The swine isolate, A/SW/OH/511445/2007 (OH07), was evaluated in an experimental challenge and transmission study reported here. Our results indicate that the OH07 virus was pathogenic in pigs, was transmissible among pigs, and failed to cross-react with many swine H1 anti-sera. Naturally exposed pigs shed virus as early as 3 days and as long as 7 days after contact with experimentally infected pigs. This suggests there was opportunity for exposure of people handling the pigs at the fair. The molecular analysis of the OH07 isolates demonstrated that the eight gene segments were similar to those of currently circulating triple reassortant swine influenza viruses. However, numerous nucleotide changes leading to amino acid changes were demonstrated in the HA gene and throughout the genome as compared to contemporary swine viruses in the same genetic cluster. It remains unknown if any of the amino acid changes were related to the ability of this virus to infect people. The characteristics of the OH07 virus in our pig experimental model as well as the documented human transmission warrant close monitoring of the spread of this virus in pig and human populations.

Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008

Prior to the introduction of the 2009 pandemic H1N1 virus from humans into pigs, four phylogenetic clusters (α-, β-, γ- and δ) of the haemagglutinin (HA) gene from H1 influenza viruses could be found in US swine. Information regarding the antigenic relatedness of the H1 viruses was lacking due to the dynamic and variable nature of swine lineage H1. We characterized 12 H1 isolates from 2008 by using 454 genome-sequencing technology and phylogenetic analysis of all eight gene segments and by serological cross-reactivity in the haemagglutination inhibition (HI) assay. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. The gene segments from the 2009 pandemic H1N1 formed clusters separate from North American swine lineage viruses, suggesting progenitors of the pandemic virus were not present in US pigs immediately prior to 2009. Serological cross-reactivity paired with antigenic cartography demonstrated that the viruses in the different phylogenetic clusters are also antigenically divergent.